Application of sodium humate in preparation of medicine for treating obesity

Abstract

The invention discloses application of sodium humate in preparation of medicines for treating obesity, and relates to the technical field of biological medicines. The research finds that the sodium humate has the effect of treating obesity, and the treatment effect on obesity can be effectively improved by further combining the sodium humate with near-infrared photothermal treatment. Through detection, the sodium humate has excellent photothermal conversion performance and good photothermal stability, and the characteristic lays a key foundation for treating obesity by combining the sodium humate with near-infrared photothermal. Experiments prove that the sodium humate can efficiently realize photothermal conversion in a fat area, and a precise action mode is provided for local fat intervention. Animal experiments prove that the sodium humate and the sodium humate combined photothermal therapy can effectively relieve the obesity degree. Meanwhile, the combined treatment can improve adipose tissue distribution, realizes fat phenotype remodeling, and especially can promote white fat browning.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of sodium humate in the preparation of drugs for treating obesity. Background Technology

[0002] Obesity is a complex metabolic disease characterized by the deposition of excess energy as triglycerides in white adipose tissue, and its prevalence has been rising in recent years. Obesity not only affects an individual's physical health but also often leads to various systemic metabolic disorders such as insulin resistance and cardiovascular disease, further increasing the risk of diseases such as diabetes, hypertension, and non-alcoholic fatty liver disease.

[0003] Despite extensive research into the pathological mechanisms of obesity, significant limitations remain in treatment options. Currently, the most common interventions are lifestyle changes, such as diet control and exercise. However, these methods heavily rely on patient self-control, and their effectiveness is often difficult to maintain. Invasive procedures for severely obese patients, such as gastrectomy, gastric bypass surgery, and adipose tissue removal, while providing significant weight loss, are accompanied by risks of infection, postoperative complications, and malabsorption, limiting their widespread application. In terms of drug therapy, existing anti-obesity medications mainly include pancreatic lipase inhibitors (such as orlistat), GLP-1 receptor agonists (such as liraglutide, benaglutide, and smegglutide), and dual GIP / GLP-1 receptor agonists (such as telpolide). These drugs can achieve short-term weight loss to some extent, but they still have many drawbacks, such as common gastrointestinal side effects (such as nausea and diarrhea), potential pancreatitis risks, and weight rebound after discontinuation, affecting patient adherence and long-term efficacy.

[0004] Therefore, there is an urgent need in the field of obesity treatment to develop safer, more effective, and sustainable new treatment strategies to achieve effective control and management of obesity and its related metabolic disorders, and to provide patients with better clinical solutions. Summary of the Invention

[0005] The purpose of this invention is to provide the application of sodium humate in the preparation of drugs for treating obesity, thereby solving the problems existing in the prior art. This invention has found that sodium humate (HA) has a therapeutic effect on obesity, and further combining it with near-infrared (NIR) photothermal therapy can effectively improve the therapeutic effect on obesity.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of sodium humate in the preparation of a medicament for treating obesity.

[0008] Furthermore, the drug is a photothermal conversion drug based on photothermal therapy.

[0009] Furthermore, the drug treats obesity by reducing body weight, lowering body fat percentage, improving fat distribution, and / or browning white fat.

[0010] This invention also provides the use of sodium humate in the preparation of a medicament for reducing liver damage caused by a high-fat diet.

[0011] Furthermore, the drug is a photothermal conversion drug based on photothermal therapy.

[0012] The present invention discloses the following technical effects:

[0013] This invention has discovered that sodium humate (HA) has a therapeutic effect on obesity, and further combination with near-infrared photothermal therapy can effectively improve the treatment effect on obesity. Testing revealed that HA possesses excellent photothermal conversion performance and good photothermal stability, a key characteristic that lays a crucial foundation for its combination with near-infrared photothermal therapy for obesity. Experiments have confirmed that HA can efficiently achieve photothermal conversion in adipose regions, providing a precise mechanism for local fat intervention. Animal experiments have confirmed that HA and HA combined with photothermal therapy can effectively reduce the degree of obesity. Simultaneously, this combined treatment can improve adipose tissue distribution and achieve adipose phenotype remodeling, especially promoting the browning of white adipose tissue—in cell experiments, HA treatment significantly reduced the size of lipid droplets within adipocytes, resulting in a brown-like adipocyte morphology. Furthermore, HA combined with NIR treatment increased the expression levels of thermogenesis-related proteins UCP1 and PGC-1α in mature white adipocytes, verifying its role in promoting the browning of white adipose tissue and enhancing thermogenic function at the cellular level.

[0014] Furthermore, this invention reveals that HA exhibits excellent safety while exerting its therapeutic effects. HA and HA combined with photothermal therapy can reduce liver damage caused by a high-fat diet, without significant damage to major tissues and organs, and without significant toxicity to adipose-derived mesenchymal stem cells (ADSCs), demonstrating good biocompatibility and safety.

[0015] In summary, HA combined with photothermal therapy achieves multiple benefits, including weight reduction, lower body fat percentage, improved fat distribution, and browning of white adipose tissue, through the photothermal effect targeting adipose areas. At the same time, it ensures the safety of major organs, providing a highly efficient and safe new approach for the treatment of obesity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Images show the speciation of sodium humate; where A: HA powder image; B: HA solutions dissolved in different media; C: HA solutions in different media after 60 days.

[0018] Figure 2 Particle size analysis diagram of HA solution;

[0019] Figure 3 The zeta potential diagram for HA;

[0020] Figure 4 The Fourier transform infrared (FLIR) spectrum of HA;

[0021] Figure 5 Vis-NIR absorption spectra of HA solutions at different concentrations;

[0022] Figure 6 To investigate the near-infrared (808 nm, 0.5 W / cm) spectra of HA solutions of different concentrations. 2 The results of the photothermal conversion performance test under irradiation are shown in the figure.

[0023] Figure 7 The heating / cooling curves of a 0.5 mg / mL HA solution after three repeated NIR irradiations are shown.

[0024] Figure 8 Figure 1 shows the experimental results of using sodium humate combined with photothermal therapy to treat obesity induced by a high-fat diet in mice. A: Flowchart of the animal experiment; B and C: Near-infrared (808 nm, 0.5 W / cm²) thermal imaging of obese mice after injection of PBS buffer or HA solution into the subcutaneous white adipose tissue in the groin area. 2 D: Thermal imaging of local temperature in mice irradiated for 180 s (B) and line graph of temperature change (∆T) (C); E: Comparison of body size of mice in each group after treatment; F: Change in body weight of mice during treatment; G: Body fat percentage of mice in each group after treatment; H: Comparison of size (G) and weight (H) of inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) among mice in each group after treatment; n = 3, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0025] Figure 9 HE staining of mouse adipose tissue in an experiment using sodium humate combined with photothermal therapy to treat obesity induced by a high-fat diet.

[0026] Figure 10 Figure 1 shows the results of a study on the molecular mechanism of sodium humate combined with photothermal therapy in treating high-fat diet-induced obese mice (DIO). A: Heatmaps of protein expression in inguinal white adipose tissue (iWAT) of each group of mice, constructed using a hierarchical clustering algorithm; B: GOBP enrichment analysis of differentially expressed proteins between mice treated with sodium humate combined with photothermal therapy and untreated obese mice; C: Western blotting results of browning indicators (UCP1 and PGC-1α) of iWAT, eWAT, and BAT; D: Immunohistochemical detection results of UCP1 in adipose tissue.

[0027] Figure 11 Figures show the liver damage and tissue compatibility test results of mice in each group after sodium humate combined with photothermal therapy; where A and B are the serum ALT (A) and AST (B) levels of mice; C is the HE staining image of the major organs and tissues (heart, liver, lung, spleen, and kidney) of mice; n = 3, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001;

[0028] Figure 12 Figures show the results of a study on the cytotoxicity of sodium humate combined with photothermal therapy and its effects on the metabolism and differentiation of mature white adipocytes; where A: Flowchart of cell experiments; B: Images of adipose-derived mesenchymal stem cells (ADSCs) before (b1) and after (b2) differentiation; C: Results of cell viability detection of ADSCs under different concentrations of HA±NIR; D: Results of lipid droplet accumulation in mature white adipocytes; E: Results of mitochondrial generation detection in different treatment groups; F: Results of protein expression detection in different treatment groups. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Example 1

[0035] I. Characterization and photothermal properties of sodium humate (HA)

[0036] 1. Preparation of HA solution

[0037] Weigh HA powder using a precision electronic analytical balance. Figure 1 Dissolve (A) in 1×PBS buffer to prepare a 5 mg / mL HA solution. Centrifuge at 1000 rpm for 3 min at room temperature to remove large, insoluble particles. After autoclaving, store at 4℃ for later use.

[0038] 2. Stability analysis of HA solution

[0039] Sodium humate powder was dissolved in different media (pure water, PBS, DMEM-F12 medium), and its state immediately after dissolution and after 60 days was photographed and recorded. Sodium humate is soluble in a variety of aqueous media and has good stability. Figure 1 (B and C in the middle).

[0040] 3. Nanoparticle Tracking Analyzer (NTA) analyzes HA particle size and quantity.

[0041] (1) Take 30 μL of HA solution (10 mg / mL) and add 15 mL of pure water to dilute it 500 times.

[0042] (2) Samples are fed into a nanoparticle tracking analyzer, and the average velocity of the particles is measured using the Stokes-Einstein equation to estimate the particle size and quantity. The particle size of most nanoparticles is between 100-150 nm. Figure 2 ).

[0043] 4. Zeta potential

[0044] (1) Take 200 μL of HA solution (10 mg / mL) and add 1 mL of pure water to dilute it 6 times.

[0045] (2) The zeta potential of the sample was detected on the instrument. The HA solution particles were negatively charged, and the potential was around -45 mV. Figure 3 ).

[0046] 5. Fourier transform infrared spectroscopy

[0047] (1) In a dry environment at room temperature, add dry HA powder and an appropriate amount of potassium bromide powder into a mortar and grind thoroughly several times. Then put it into a tablet press and press it into a transparent sheet. Finally, test it on the machine.

[0048] (2) Before testing, the background was collected to remove background noise, and then the infrared spectrum of the sample was collected with a resolution of 4 cm⁻¹. -1 The number of scans was 32, and the test band was 400-4000 cm. -1 The chemical structure of HA contains active functional groups such as carboxyl, quinone, and phenolic hydroxyl groups. Figure 4 ).

[0049] 6. Visible-Near Infrared Absorption Spectrum

[0050] (1) Dissolve HA powder in pure water to prepare HA solutions of different concentrations (0, 0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL), and add 200 μL of each solution to a 96-well plate.

[0051] (2) The absorbance at 600 nm-900 nm was detected using a SpectraMax iD3 multi-mode microplate reader. Compared with pure water, HA exhibits a broad absorption spectrum in the near-infrared region. Figure 5 ).

[0052] 7. Determination of the in vitro photothermal properties and stability of HA solution

[0053] (1) Prepare 1 mL of HA solutions with concentrations of 0.05, 0.1, 0.25, 0.5, 1, and 2 mg / mL and place them in a 1.5 mL EP tube.

[0054] (2) Fix the collimator (10 mm in diameter) of the fiber-coupled laser (808 nm) at a height of 10 cm above the liquid surface using an iron stand. Set the current to achieve an output power of 0.5 W / cm². 2 The liquid was irradiated, and temperature changes were detected using a FLIR infrared thermal imager, recording the temperature changes every 30 seconds over 3 minutes. Compared to pure water, the HA solution exhibited higher temperature changes after NIR irradiation, and these changes showed concentration- and time-dependent characteristics. Figure 6 This demonstrates that HA possesses excellent photothermal conversion performance.

[0055] (3) Using 0.05 mg / mL as the determination concentration, the infrared laser was first turned on to measure the temperature rise of the liquid over three minutes, recording the temperature every 30 seconds. After three minutes, the infrared light was removed, and the temperature change after removal was recorded. The temperature was recorded every 30 seconds for the first 10 minutes, and then every minute thereafter, until the temperature dropped to 24°C. This process was repeated twice, and the temperature changes were recorded to create a cold-heat cycle curve. After three repeated heating / cooling cycles, the photothermal conversion property of the HA solution was not reduced ( Figure 7 This demonstrates that HA has good photothermal stability.

[0056] II. Photothermal effect of in situ injection of HA into white adipose tissue in the groin of mice

[0057] (1) Take a sterile insulin syringe, draw 100 μL of PBS or HA solution, and inject it into the groin area of ​​the mouse parallel to the ventral surface.

[0058] (2) After checking that there are no problems, put the mouse back in the cage.

[0059] (3) After 24 h, the fiber collimator of the infrared laser was fixed at a height of 10 cm from the groin area of ​​the mouse, and NIR (808 nm, 0.5 W / cm) was applied. 2 Irradiation was performed using an infrared thermal imager, and temperature changes were recorded every 30 seconds. The results are shown in […]. Figure 8 In mice injected with HA, the temperature of the subcutaneous white fat region in the groin was significantly increased after NIR irradiation.

[0060] III. Study on the Efficacy and Molecular Mechanism of HA Combined with Photothermal Therapy for High-Fat Diet-Induced Obesity in Mice

[0061] 1. Construction and treatment of mouse models

[0062] Thirty male SPF-grade C57BL / 6J mice, aged 6-8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization in the animal facility, they were divided into five groups (NCD, HFD, HFD+HA, HFD+NIR, and HFD+HA+NIR), with six mice in each group. The NCD group was fed a 10 kcal% fat-based diet (XTCON50J from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.), while the HFD, HFD+HA, HFD+NIR, and HFD+HA+NIR groups were fed a 60 kcal% fat-based diet (XTHF60 from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.). Body weight was recorded weekly. After 10 weeks of feeding, treatment began, with the treatment methods for each group as follows: Figure 8 As shown in A and Table 1.

[0063] Table 1 Experimental Groups

[0064]

[0065] The body weight changes of each treatment group before and after each cycle were recorded, and the treatment lasted for seven cycles. After seven cycles of treatment, the body weight of mice in the HFD+HA+NIR group showed a significant decreasing trend. Figure 8 (E).

[0066] 2. Measurement of mouse body fat using the EchoMRI-100H animal body composition analyzer

[0067] Body fat percentage in mice of each treatment group was measured using an EchoMRI-100H animal body composition analyzer. Results showed that the body fat percentage in the HA combined with photothermal therapy group was significantly reduced, approaching that of the NCD (normal control) group. Figure 8 (Middle F).

[0068] 3. Collection of mouse serum, adipose tissue, and major organs

[0069] (1) First, anesthetize the mice, weigh the mice, and calculate the dosage of anesthetic. The dosage of 1% sodium pentobarbital is 8 μL / g.

[0070] (2) Using the same steel ruler as a reference, place the mouse on a piece of white paper and take a rough picture. Figure 8 (D). Mice in the HFD group were more rounded and shorter, while mice treated with HA combined with photothermal therapy were more slender.

[0071] (3) The mice were then dissected. After exposing the abdominal cavity, the pleura was cut open, and blood from the heart was drawn using a clean 1 mL syringe. The needle was removed, and the blood was injected into a 1.5 mL EP tube. After standing at room temperature for 1 h, the tube was centrifuged (4℃, 3000 rpm, 10 min). The separated serum could be temporarily stored at -80℃. The inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), brown adipose tissue (BAT), and major organs (heart, liver, lung, spleen, and kidney) of the mice were removed with scissors and forceps and placed in PBS buffer on ice. The organs were fixed or stored at -80℃ for later use, depending on the experimental requirements.

[0072] (4) Place the separated iWAT, eWAT and BAT on the membrane and take a picture to record the results. Figure 8 (G). Record the weight of the three types of fat in each group of mice and calculate their ratio to body weight (G). Figure 8 The results showed that the volume and weight ratio of white adipose tissue increased in the HFD group, but recovered after HA combined with photothermal therapy, approaching that of normal mice; the volume and weight ratio of brown adipose tissue decreased in the HFD group, but increased after HA combined with photothermal therapy, proving that HA combined with photothermal therapy can improve adipose tissue distribution.

[0073] 4. Hematoxylin-eosin (HE) staining of adipose tissue

[0074] Tissue section preparation and HE staining were outsourced to Hunan Aifang Biotechnology Co., Ltd., and the specific steps are as follows:

[0075] (1) Sampling and fixation: Fresh mouse tissue was rinsed with pre-cooled PBS buffer to remove blood and impurities. A small piece was cut off and immediately placed in 4% paraformaldehyde fixative and fixed at room temperature for more than 24 hours.

[0076] (2) Perform tissue embedding according to the steps shown in Table 2:

[0077] Table 2 Specific procedures for tissue embedding

[0078]

[0079] (3) Paraffin sectioning: Place the wax block on a paraffin microtome and section it to a thickness of 4 μm. Float the section on 40℃ warm water in a slide spreader to flatten the tissue. Pick up the tissue with a glass slide and bake it in a 65℃ oven. After the wax has melted in the water, remove it and store it at room temperature for later use.

[0080] (4) Dewaxing and hydration: xylene, twice, 10 min each time; anhydrous ethanol → 95% ethanol → 85% ethanol → 75% ethanol → distilled water, 5 min per stage.

[0081] (4) HE staining: Add hematoxylin staining solution to cover the tissue and stain for 5 min → rinse with running water to remove the floating color → differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with running water → return to blue with 1% ammonia water solution for 1 min, rinse with running water → eosin staining solution for 2 min, and then wash away the floating color with distilled water.

[0082] (5) Dehydration and mounting: 75% ethanol, 85% ethanol, 95% ethanol and 100% ethanol for 1 min each → xylene, 1 min → xylene, 1 min → neutral resin for mounting.

[0083] (6) Microscopic observation and image acquisition and analysis. The results showed that both white adipose tissue and brown adipocytes in the HFD group were enlarged, while adipocytes decreased and returned to normal morphology after HA combined with photothermal therapy. Among them, iWAT showed multilocular adipocytes and "white adipose tissue turning brown" morphological changes, proving that HA combined with photothermal therapy can achieve adipose phenotype remodeling in diet-induced obese mice. Figure 9 ).

[0084] 5. Extraction of total protein from tissues

[0085] (1) Prepare rapid cell lysis buffer (RIPA) and protease inhibitor benzyl sulfonyl fluoride (PMSF), vortex mix them at a volume-to-mass ratio of 100:1 and place them on ice.

[0086] (2) Take the tissue sample out of the -80℃ freezer and put it into a 1.5 mL EP tube. Add the corresponding lysis buffer at a ratio of tissue weight: lysis buffer = 1:9 (g:mL).

[0087] (3) Grind the tissue with a grinder and lyse it on ice for 30 min.

[0088] (4) Pre-cool the centrifuge at 4°C, centrifuge at 14,000 rpm for 30 min; collect the supernatant after centrifugation; adipose tissue needs to be centrifuged twice to remove the upper layer of fat.

[0089] (5) The supernatant was used for BCA protein quantification and was processed differently according to the experimental purpose.

[0090] 6. Mouse iWAT proteomics analysis

[0091] (1) Sample preparation and standardization: The protein samples extracted from the white adipose tissue of the groin were randomly selected from each group; after quantification by BCA method, the concentration was uniformly adjusted to 1 μg / μL with lysis buffer, and 100 μg / sample was tested (samples were sent on ice).

[0092] (2) Mass spectrometry detection: The samples were sent to the large-scale instrument platform of Wenzhou Medical University. After enzymatic digestion and liquid chromatography separation, they were detected by the instrument. The original protein spectrum identification data were obtained by using a quadrupole-electrostatic field orbital trap-linear ion trap triplet (Orbitrap Fusion Lumos) ultra-high resolution mass spectrometer.

[0093] (3) Data quality control and filtering: The corresponding protein sequence number was searched in the UniProt database. Strict quality control standards were applied according to Table 3.

[0094] Table 3 Data Quality Control

[0095]

[0096] A total of 2,808 reliable proteins were ultimately selected for further analysis.

[0097] (4) Hierarchical clustering of basic protein expression: Heatmaps were generated using a hierarchical clustering algorithm on the BioLadder online bioanalysis platform. The protein expression patterns of each group of samples exhibited significant grouping characteristics. Figure 10 (A). Among them, the overall adipose tissue proteomics characteristics of the HFD+HA group and the HFD+HA+NIR group showed a high correlation, while the proteomics characteristics of the HFD group and the HFD+NIR group were highly similar, suggesting that single NIR intervention may be difficult to reverse the protein expression abnormalities induced by a high-fat diet.

[0098] (5) Bioinformatics analysis: 1403 differentially expressed proteins with significant differences (p < 0.05, fold change > 2) were screened. GO analysis was performed using the DAVID database, revealing that these proteins were mainly enriched in lipid metabolism-related biological processes, specifically promoting brown adipocyte differentiation (e.g., UCP1) expression changes. Figure 10 (B)

[0099] 7. Western Blotting

[0100] (1) Protein sample preparation: After BCA quantification, add 5× loading buffer and lysis buffer to adjust to the same concentration, denature in a metal bath at 100℃ for 5 min, and then incubate briefly. It can be stored at -30℃;

[0101] (2) Gel preparation: Prepare 10% SDS-PAGE gel;

[0102] (3) Sample loading: Fix the two SDS-PAGE gel plates back and forth with a clamp and place them in the electrophoresis tank according to the correspondence between the positive and negative electrodes. Add freshly prepared 1×SDS electrophoresis buffer diluted with ddH2O to the inner tank and add recycled electrophoresis buffer to the outer tank. Carefully remove the comb and carefully add the denatured protein sample into the sample well, marking it on both sides;

[0103] (4) Electrophoresis: Set the conditions as follows: stacking gel 70 V, 40 min; separating gel 110 V, 60-70 min. Start electrophoresis after setting.

[0104] (5) Transfer: Take out the pre-cooled 1× transfer solution and pour it into the tray. After rinsing the SDS on the gel plate with running water, immerse it in the transfer solution. Prepare the transfer clamp, with the black side down, and assemble it according to the sandwich structure "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". Note that the PVDF membrane needs to be activated with methanol; there should be no air bubbles between the layers. After clamping the transfer clamp, place it in the electrophoresis tank, paying attention to the correspondence of the positive and negative electrodes. Add the transfer solution and add ice to maintain the low temperature of the transfer solution. After confirming that everything is correct, set the temperature to 250 mA and perform the transfer for 100 min.

[0105] (6) Sealing: Prepare 5% skim milk, put the polyvinylidene fluoride (PVDF) membrane after transfer into the milk, and seal it in a shaker at room temperature for 2 h;

[0106] (7) Primary antibody incubation: After blocking, wash three times with 1×TBST shaker for 5 min each time. Cut the band according to the size of the target protein and the marker position, put it into the corresponding primary antibody, and incubate overnight at 4°C on a shaker;

[0107] (8) Incubation with secondary antibody: Take the band from the primary antibody, wash it three times with 1×TBST shaker for 5 min each time, and then put it into the corresponding secondary antibody tube and incubate it in a shaker at room temperature for 70 min.

[0108] (9) Exposure: Remove the band from the secondary antibody and wash it three times with a 1×TBST shaker for 15 min each time. Prepare ECL colorimetric solution (solution A:solution B = 1:1). After washing the band, gently blot the water with filter paper, transfer it to the exposure instrument, add the colorimetric solution to cover the band with a pipette, close the chamber door, and click exposure to acquire the image.

[0109] Imaging results showed that the expression of UCP1 and PGC-1α in the inguinal white adipose tissue of mice in the HFD+HA+NIR group was significantly higher than that in the HFD group, approaching the level of the NCD group, demonstrating that HA combined with local photothermal activity promoted the browning of white adipose tissue in obese mice, and the expression of thermogenic proteins in whole-body fat (eWAT, BAT) was also increased. Figure 10 (C)

[0110] 8. UCP1 Immunohistochemistry

[0111] Tissue section preparation and UCP1 histochemical staining were outsourced to Hunan Aifang Biotechnology Co., Ltd., and the specific steps are as follows:

[0112] Steps (1)-(4) are the same as "4. Hematoxylin-eosin (HE) staining of adipose tissue";

[0113] (5) Block endogenous peroxidase; soak tissue sections in 3% hydrogen peroxide, incubate at room temperature in the dark for 15 min, and rinse the slides twice with PBS buffer, 5 min each time;

[0114] (6) Antigen retrieval: Tissue sections were placed in a retrieval box filled with EDTA antigen retrieval buffer (final concentration of 0.01 mol / L), heated in a microwave oven for 10 min on medium heat, 5 min off heat, 5 min on medium-low heat, cooled to room temperature, and washed twice with PBS buffer on a shaker for 5 min each time.

[0115] (7) Blocking: Add 100 μL of non-immune normal goat serum to cover the tissue (draw a chemical circle at the edge), incubate at room temperature for 30 min, and then aspirate;

[0116] (8) Primary antibody incubation: Add 1:400 diluted UCP1 primary antibody to cover the tissue, place in a humidified box and incubate overnight at 4°C, then wash with PBST for 5 min each time;

[0117] (9) Secondary antibody incubation: Add anti-rabbit antibody to cover the tissue, incubate at room temperature for 30 min, and wash with PBS three times with shaking, 5 min each time;

[0118] (10) DAB reaction color development: Add DAB color development solution (solution A:solution B = 50:1) to the histochemistry zone, observe the color of the section under the microscope, and immediately rinse with tap water to stop the color development after it turns brownish-yellow.

[0119] (11) Counterstaining: Counterstain with hematoxylin for 1 min, then rinse with running water for 2 min;

[0120] (12) Differentiation and blueing: Differentiate with 1% hydrochloric acid alcohol for 2 seconds, then rinse with running water for 15 minutes;

[0121] (13) Dehydration and clearing: 85%, 95%, anhydrous ethanol, and anhydrous ethanol, 5 min each → dewaxing twice in xylene, 5 min each time;

[0122] (14) After natural drying, the slides were sealed with neutral resin, observed under a microscope, and images were collected.

[0123] The results showed that the density of UCP1-positive cells in the adipose tissue of the HFD group was reduced, but UCP1 expression increased after HA combined with photothermal therapy. Figure 10 (D) further demonstrates that HA combined with photothermal activity can promote the browning of white adipose tissue.

[0124] IV. Liver injury and tissue compatibility test in mice

[0125] 1. Serum alanine aminotransferase (ALT) detection

[0126] (1) Take a clean 96-well transparent plate and add samples according to Table 4:

[0127] Table 4 ALT Detection Sample Dosing Table

[0128]

[0129] (2) After incubation for 30 min in step (1), a standard curve was prepared, and samples were added according to Table 5:

[0130] Table 5. Sample Addition Table for Preparing the ALT Standard Curve

[0131]

[0132] A fitting curve was plotted with the absolute OD value (OD value of each well minus the OD value of the zero well) on the x-axis and enzyme activity on the y-axis. Substituting the absolute OD values ​​of the above samples into the standard curve yielded their ALT enzyme activity.

[0133] 2. Serum aspartate aminotransferase (AST) detection

[0134] (1) Take a clean 96-well transparent plate and add samples according to Table 6:

[0135] Table 6 AST Detection Sample Addition

[0136]

[0137] Calculate the absolute OD value: OD value of the test well - OD value of the control well.

[0138] (2) After incubation for 30 min in step (1), a standard curve was prepared simultaneously, and samples were added according to Table 7:

[0139] Table 7. Sample Addition Table for Preparing the AST Standard Curve

[0140]

[0141] A fitting curve was plotted with the absolute OD value (OD value of each well minus the OD value of the zero well) on the x-axis and enzyme activity on the y-axis. Substituting the absolute OD values ​​of the above samples into the standard curve yielded their AST enzyme activity.

[0142] 3. HE staining of major organs

[0143] The procedure is the same as in "III.4. Hematoxylin-eosin staining of adipose tissue". HE staining of the liver showed that in normal livers, hepatocytes were arranged in an orderly manner, and the lobular structure was clear; while in the HFD group and the HFD+NIR group, the livers had numerous fat vacuoles and disordered lobular structure; after HA treatment, the above pathological changes in both groups decreased, and the liver morphology returned to normal. No obvious lesions were observed in the major organs, indicating that HA and / or NIR irradiation does not cause damage to the major tissues and organs of mice. Figure 11 (C)

[0144] The results of ALT and AST detection in the serum of mice in each treatment group are shown below. Figure 11 A and B in the middle, combined with liver HE staining ( Figure 11 The results showed that serum ALT and AST levels were elevated in the HFD group mice, confirming that a high-fat diet leads to liver damage, while HA and HA combined with photothermal therapy can reduce these levels and reduce liver damage.

[0145] V. Extraction and Differentiation of Mouse Adipose-Derived Mesenchymal Stem Cells (ADSCs)

[0146] 1. Primary isolation and extraction of ADSCs

[0147] (1) Anesthetize mice with 8 μL / g of anesthetic, and after they are completely unconscious, disinfect them by soaking them in 75% ethanol for 15 min and then place them in a clean bench.

[0148] (2) To avoid contamination from mouse hair, cut the bilateral inguinal fat in parallel.

[0149] (3) Wash the inguinal adipose tissue repeatedly three times with PBS buffer containing 1% penicillin-streptomycin solution (100×), and then wash it three times with PBS buffer.

[0150] (4) Under the microscope, remove blood vessels, lymph nodes, connective tissue, etc. as thoroughly as possible, and cut them into pieces of about 1 cm. 3 Small pieces.

[0151] (5) Transfer to 2 times the volume of 0.2% collagenase Digestion was performed in a 37°C constant temperature shaking incubator in the solution. After 30 min, centrifugation was carried out at 1000 rpm for 5 min at room temperature. The cell pellet was collected and resuspended in DMEM-F12 complete medium. The remaining supernatant was further digested for 15 min, and the cycle was repeated twice until the tissue appeared cloudy or milky.

[0152] (6) Add an equal amount of DMEM-F12 complete culture medium to stop digestion, filter through a 70 μm cell filter to remove undigested tissue.

[0153] (7) Centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant. Wash the precipitate with PBS buffer, centrifuge at 1000 rpm for 5 min, discard the supernatant, and repeat twice;

[0154] (8) Add red blood cell lysis buffer, lyse at 37°C for 5 min, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0155] (9) Gently pipette the cells into a single-cell suspension using DMEM-F12 complete medium and inoculate them into culture dishes. Incubate in an incubator (conditions set to 37℃, 5% CO2). Change the medium every other day.

[0156] 2. ADSCs cell differentiation induction

[0157] like Figure 12 As shown in Figure A, the following cell experiments were performed:

[0158] (1) Cell seeding and contact inhibition: with 5×10 3 cells / cm 2 The cells were seeded into culture plates at a certain density, and their growth status was monitored daily. The cells were cultured for another 24 hours while maintaining contact inhibition (recorded as day 0).

[0159] (2) Discard the original culture medium and replace it with a preheated lipogenesis induction culture medium at 37°C. Place the medium in an incubator and incubate for three days. Observe the changes in cell morphology during the treatment period.

[0160] (3) On the third day, the lipid induction medium was removed and replaced with an equal volume of lipid maintenance medium, and the medium was placed back in the incubator for four days of induction.

[0161] (4) On day 7, discard the adipogenic maintenance medium and replace it with ordinary DMEM-F12 complete medium. Observe the differentiation of adipocytes daily under an inverted microscope, changing the medium every two days until the differentiation rate reaches more than 80%. Take photos to record the morphology of cells before and after induction. Figure 12 (B) Before induction, ADSCs are spindle-shaped or fibroblast-like, and mature adipocytes induced by the "cocktail method" exhibit large and clear lipid droplets.

[0162] VI. Cytotoxicity (MTT) Detection

[0163] Using the standard MTT assay and ADSCs cells as a model, we investigated the cytotoxicity of HA±NIR ("±" indicates combined or uncombined NIR, where uncombined NIR is natural light).

[0164] (1) First, perform cell plating. Plating requirements: 96-well plate, 3000 cells / well, 60 wells, plus 5 control wells without cells, 100 μL of system in each well.

[0165] (2) After 12 h of cell adhesion, the 96-well plate was removed and the cell state in the well was observed under a microscope. Then the original culture medium in the experimental wells was discarded, and HA solution (diluted with DMEM-F12 complete culture medium) with a final concentration of 0, 25, 50, 75 and 100 μg / mL was added respectively. Finally, the plate was put back into the incubator for 24 h.

[0166] (3) After 24 hours, remove the 96-well plate and expose it to white light or NIR (808 nm, 0.5 W / cm²) respectively. 2 After treatment (10 min), the cells were incubated in the incubator for another 12 h before proceeding to the next step.

[0167] (4) Take out the 96-well plate, replace the original culture medium with DMEM-F12 blank culture medium containing 0.5 μM diphenyltetrazolium bromide (MTT), and then put it in an incubator to continue incubation for 4 h.

[0168] (5) Next, add 100 μL of formazan solution directly to each well, and then dissolve the formazan in a 37°C incubator.

[0169] (6) After the formazan has completely dissolved, use a SpectraMax iD3 multi-microplate reader to measure the absorbance (OD value, expressed as A) of each well at 570 nm and calculate the cell viability using the following formula:

[0170] Cell viability (%) = (A 样本孔 - A 对照孔 ) / (A 空白孔 - A 对照孔 ) × 100%.

[0171] The results showed that, regardless of whether 808 nm laser irradiation was applied, HA concentrations of 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL had no significant toxicity to ADSCs cells. Figure 12 (C)

[0172] VII. Effects of HA combined with photothermal therapy on the metabolism and differentiation of mature white adipocytes

[0173] 1. Oil Red O staining of adipocytes

[0174] (1) When the adipocyte differentiation rate is ≥80%, replace the conventional DMEM-F12 complete medium with solutions of HA final concentrations of 0, 50, and 100 μg / mL (prepared with DMEM-F12 complete medium) and incubate for 24 h. Figure 12 (A)

[0175] (2) Half of the light was treated with white light, and the other half was treated with NIR (808 nm, 0.5 W / cm²). 2 After treatment (10 min), the cells were placed in an incubator and cultured for another 12 h.

[0176] (3) Remove the cell plate after 12 h, discard the old culture medium, wash 3 times with PBS buffer, gently shake to prevent cells from detaching from the cell wall, and discard the PBS.

[0177] (4) Add 4% paraformaldehyde to each well and fix at room temperature for 30 min.

[0178] (5) Discard the fixative, wash 3 times with PBS buffer, and discard.

[0179] (6) Add 60% isopropanol solution along the side wall and mordant for 3 min.

[0180] (7) Remove isopropanol, add freshly prepared Oil Red O working solution along the side wall, and stain at room temperature in the dark for 30 min.

[0181] (8) Discard the Oil Red O working solution and wash away the residual dye with PBS buffer until the upper layer of solution is colorless.

[0182] (9) Observe the lipid droplet deposition in the cells using an inverted optical microscope and take photos to record the data.

[0183] Mature adipocytes induced by the "cocktail method" exhibit large and translucent lipid droplets. After HA treatment, the size of the lipid droplets within the adipocytes is significantly reduced, and they exhibit a more brownish adipocyte morphology. Figure 12 (D).

[0184] 2. Mitochondrial Deep Red fluorescent probe staining

[0185] (1) Prepare preheated serum-free DMEM-F12 medium and dilute the probe at 1:1000.

[0186] (4) Remove the old culture medium and gently wash the cells with PBS buffer to remove any residual serum-containing culture medium.

[0187] (5) Add 300 μL of serum-free culture medium containing the probe to each well and incubate at 37°C for 30 min.

[0188] (6) Discard the staining solution, wash three times with PBS buffer, and add 300 μL of serum-free culture medium.

[0189] (7) Observe under an inverted fluorescence microscope.

[0190] Note: Keep the entire process away from light to avoid fluorescence quenching, which would affect the staining effect.

[0191] Fluorescence microscopy revealed a significant increase in the number of mitochondria in mature adipocytes treated with HA+NIR. Figure 12 (E).

[0192] 3. Protein extraction and Western blotting from mature adipocytes

[0193] (1) Discard the original culture medium and wash with PBS 3 times.

[0194] (2) Add 60 μL of lysis buffer to each well, scrape it off with a cell scraper, and collect it into a 1.5 mL EP tube.

[0195] (3) After lysing on ice for 30 min, centrifuge at 14000 rpm for 30 min at 4℃, and collect the supernatant after centrifugation. The protein concentration was adjusted to 3 mg / mL by BCA method and denatured at 100℃ for 5 min.

[0196] (4) See “III.7. Protein Blotting” for the steps of Western Blotting.

[0197] Figure 12 The results showed that combined treatment with HA and NIR increased the expression levels of the thermogenic proteins UCP1 and PGC-1α in mature white adipocytes, and also verified at the cellular level that HA combined with local photothermal therapy can promote the browning of white adipocytes.

[0198] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of sodium humate in the preparation of a medicament for the treatment of obesity.

2. Use according to claim 1, characterized in that, The medicament is a photothermal conversion medicament based on photothermal therapy.

3. Use according to claim 1, characterized in that, The medicament treats obesity by reducing body weight, lowering body fat percentage, improving fat distribution, and / or browning white adipose tissue.

4. Use of sodium humate in the preparation of a medicament for reducing liver damage caused by a high-fat diet.

5. Use according to claim 4, characterized in that, The medicament is a photothermal conversion medicament based on photothermal therapy.

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